WO1995034579A1

WO1995034579A1 - Recombinant molecules, proteins/polypeptides, host systems and diagnostic and therapeutic methods for atopy

Info

Publication number: WO1995034579A1
Application number: PCT/SE1995/000723
Authority: WO
Inventors: Rudolf Valenta; Susanne Natter; Susanne Seiberler; Peter Valent; Dietrich Kraft
Original assignee: Pharmacia Ab
Priority date: 1994-06-14
Filing date: 1995-06-14
Publication date: 1995-12-21
Also published as: AU2759795A; SE9402090D0

Abstract

Recombinant DNA molecules comprising a nucleotide sequence which codes for the atopy related antigens Ka, Kb, ara-3 or ara-4 (as defined in figures 1 - 4) or homologous forms thereof or at least one epitope thereof, or a nucleotide sequence which hybridizes with such a sequence under conditions of high stringency. Recombinant DNA expression vectors and host cells comprising these DNA molecules. Polypeptides encoded by the recombinant DNA molecules. Diagnostic and therapeutic methods employing an atopy related antigen, in particular the Ka, Kb, ara-3, ara-4 and ara-5 antigens that are based on the sequences defined in figures 1 - 5.

Description

RECOMBINANT MOLECULES, PROTENS/POLYPEPTIDES, HOST SYSTEMS AND DIAGNOSTIC AND THERAPEUTIC METHODS FOR ATOPY.

INTRODUCTION

Atopic dermatitis represents an itchy cutaneous disease which is mostly associated with a high level of total serum IgE and a pronounced TH-2 cellular immune response (1-5). It has been shown that in early atopic dermatitis lesions vascular hypertrophy, endothelial cell activation and skin infiltration with mast cells, basophils, eosinophils and T-cells occurs (6). In addition Langerhans cells and monocytes of atopic patients were found to express increased amounts of the high affinity receptor for IgE (7-8). T-cells obtained from atopic dermatitis patients mostly belong to the TH-2 subset secreting more IL-4 whereas interferon- γ levels were found to be reduced in atopic dermatitis (9-14). It can thus be assumed that atopic dermatitis such as Type I allergy belongs to diseases with a TH-2 activation.

In Type I allergy the environmental allergens (pollen proteins, mite proteins...) can be clearly defined (15,16) and the

specificity of IgE-antibodies against environmental allergens significantly correlates with the course of the disease. In the case of atopic dermatitis the high levels of serum IgE do not correlate with a certain sensitization pattern and no specific antigen spectrum could be defined as yet. Although it has been shown that atopic dermatitis patients display IgE-reactivity to various mite, pollen and food allergens (17) no clear association between a certain allergen source and the disease could be established.

Based on the notion of a similarity between a birch pollen allergen identified as the cytoskeletal protein, profilin and human profilin (2) we investigated whether certain atopic diseases might be associated with autoreactivity against human proteins. Despite the ability of plant and mammalian profilins to interact equally well with plant and mammalian actins thus suggesting a high degree of structural similarity of different profilins (27,20) no direct evidence could be provided that IgE- autoreactivity against profilins can trigger severe symptoms of atopy. Suggestions about the involvement of profilin as an autoantigen in allergic diseases and atopy have also been put forward in the patent litterature (35). Neither in this latter publication any real substantiation of the idea was presented.

During the priority year the results presented herein was partially published for the Ka antigen (12th European Immunology Meeting, June 14-17, Barcelona, European Federation of

Immunological Science).

Definitions

If not otherwise specified, the expressions "Ka antigen" and "Kb antigen" mean any peptide/protein in which the amino acid sequences (minus underlined parts) given in figure 1 and 2, respectively, or parts thereof unique for Ka and Kb can be retrieved. For example the complete native forms and genetic engineered variants containing different combinations of one or more unique parts/epitopes of the complete native forms.

By the expression "a polypeptide displaying the antigenicity of the Ka or Kb antigen" is meant any peptide displaying antigenic crossreactivity with one or more epitopes that are unique for the Ka or Kb antigen. One can check for crossreactivity by inhibition experiments.

Preparations of the Ka or Kb antigen are normally substantially pure in the sense that their content of other proteins/peptides originating from the source in which they have been produced normally is < 50 % such as < 25 % or < 10 % or < 1% (w/w).

During the priority year the IgE reactive autoantigens of the type concerned herein have been named atopy related antigens (ara). Ara-1 is the Kb and ara-2 the Ka antigen. The

definitions/statements concerning Ka and Kb are valid also for the atopy related antigens (ara-3, ara-4 and ara-5), the cDNA of which are partially presented in figures 3-5. The term

autoantigen below refer to atopy related antigens, if not otherwise specified. By hybridization during high stringency is meant conditions giving essentially the same or better specificity as the

conditions applied in the experimental part for estimating the sizes of the native forms of the Ka and Kb antigens via

hybridization to the corresponding mRNAs .

The term epitope (B-cell epitope) in the context of the present invention identifies a region on an antigen where antibodies can bind, for instance IgE or IgG antibodies that may be of

mammalian, particularly human, origin. The term primarily

designates the IgE-binding site for exclusively one antibody with a given specificity.

Objectives

The objectives of the invention are to provide simple, better and more reliable in vitro an in vivo tests for atopy, in

particular atopic dermatitis, as well as therapeutic methods for this disease.

The invention

One aspect of the invention is a recombinant DΝA molecule

comprising

i) a nucleotide sequence which codes

i:1) for a polypeptide displaying the antigenicity of the atopy related antigens Ka, Kb, ara-3 or ara-4 or 1:2) for a polypeptide that is homologous to > 50 %, such as > 60 %, > 70 % or > 80 % to these atopy related antigens, or

i:3) for a peptide comprising at least one epitope unique for (a) these atopy related antigens or (b) a peptide that is homologous to > 50 %, such as > 60

%, > 70 % or > 80 % to the Ka or Kb antigen, or ii) a nucleotide sequence which hybridizes with such a

sequence under conditions of high stringency.

A second aspect of the invention is a recombinant DΝA

expression vector or cloning system comprising an expression control sequence operatively linked to a nucleotide sequence defined in (i) and (ii) above.

A third aspect of the invention is a host cell or host system containing a recombinant DNA molecule or a recombinant expression vector as defined above.

A fourth aspect of the invention is a polypeptide comprising the amino acid sequence of one of the atopy related antigens defined above or at least one epitope unique for these atopy related antigens. Preferred modes of this aspect comprise that the polypeptide have been produced recombinantly or

synthetically. Another preferred mode comprises that the

polypeptide has been derivatized in the sense that it is a) linked to a water-insoluble phase by physical adsorption or by a covalent bond, b) conjugated covalently to an analytically detectable group (label), and/or c) covalently linked to an additionally polypeptide, for instance by being recombinantly produced in the form of a fusion protein comprising the

additional protein.

The water-insoluble phase may be a polymer that is water- insoluble and selected from insoluble forms of polysaccharides and their derivatives, for instance dextran, pullulan, agarose, cellulose etc, or synthetic polymers, preferably vinyl polymers, such as polyacrylamides, polyacrylates, polystyrene, polyvinyl alcohol, polyvinyl ethers etc. The physical form of the insoluble phase may be: walls of microtitre wells, spheres, rods, sheets, strips, pads etc, said physical forms may be porous or non- porous.

The additional protein may be β-galactosidase, GST or lambda ell protein or any other polypeptide which can be expressed in a prokaryotic or eukaryotic cell.

A fifth aspect of the invention is a method for in vitro diagnosis of atopy or inflammation in a mammalian individual, such as a human individual, by detecting/determining abnormal levels (preferably elevated) of antibodies directed against an atopy related antigen. This method comprises detection of the reaction of IgE in a body fluid sample from the individual with an IgE autoantigen from the same species as the individual or an IgE hapten/autoantigen crossreacting with the IgE autoantigen, optionally derived from another source. The formation of an IgE immune complex is taken as an indication that the individual is suffering from atopy or inflammation.

The atopy preferred to diagnose at the filing date of this specification is atopic dermatitis and intrinsic asthma

bronchiale with elevated IgE.

The preferred autoantigens to be used in this aspect of the invention are the atopy related autoantigens as defined above and autoantigens comprising an epitope of ara-5, the cDNA sequence of which is given in figure 5.

The body fluid sample contains IgE is mostly derived from blood, such as a whole blood sample, a serum sample or a plasma sample. Also other fluids such as CSF, urine etc may have a potential use.

One preferred protocol of the method is to bring the

autoantigen in insoluble form or in insolubilizable form into contact with the sample under conditions permitting formation of an immune complex between the autoantigen and antibodies present in the sample, whereafter the amount of complexed IgE antibodies is detected, where appropriate after insolubilization of the formed IgE-autoantigen complex. Detection may preferably be done by the use of labeled ani-IgE antibody.

Another preferred protocol of the method employs labeled autoantigen, and the immune complex between the autoantigen and sample IgE is formed in a soluble form that in the course of being quantitated is insolubilized by being contacted with water- insoluble or water-insolubilizable anti-IgE antibodies,

preferably linked to the solid phases given above.

The above-mentioned methods including the preferred protocols may analogously be used for the determination of antibodies of other classes, e.g. IgG.

A sixth aspect of the invention is a method to measure in vitro a cellular reaction against an atopy related autoantigen

comprising the step of stimulating or inhibiting the cellular reaction with the atopy related autoantigen. Examples of suitable autoantigens contain at least one atopy related epitope present in either Ka, Kb, ara-3, ara-4 or ara-5. The stimulation/inhibition may be performed by measuring histamine liberated from basophils or mast cells which have been loaded with IgE specific for an atopy related autoantigen plus the autoantigen or with IgE complexed to the atopy related autoantigen. Measuring can also be done through proliferation of autoantigen specific T cells (for example as ³H thymidine uptake). This method too may be used for the diagnosis of atopy as defined for the fifth aspect of the invention.

A seventh aspect of the invention is a method of treatment of an mammalian individual, in particular a human being, suffering from atopy wherein an effective amount of a polypeptide

comprising an epitope of an atopy related autoantigen as defined above is administered to said individual . The same

protocols/modes as used with pollen extracts are potentially applicable. In addition one might think about clearing a

patient's plasma by affinity adsorption to an insolubilized atopy related autoantigen. All modes of therapy which are currently considered for humoral autoimmune diseases (tolerance induction etc) may also be applied to those patients who display IgE- autoreactivity.

An eighth aspect of the invention is a method to diagnose inflammation or atopy by detecting/determining the prescence of abnormal levels (normally elevated) of an IgE- or IgG-atopy related antigen as defined above (preferably related to the Ka, Kb, ara-3, ara-4 or ara-5 antigens). The protocols to be employed comprise adsorbing out the atopy related antigen from a body sample by use of an antibody and detecting in a known per se mannner the antigen. The antibody may be directed against the atopy related antigen or against an antibody complexed to the atopy related antigen, for instance an anti-IgE antibody in case the antigen is complexed to IgE in the sample. At the filing of this specification, the preferred modes of all eighth aspects made use of recombinant DNA molecules (aspects 1- 3) or where appropriate polypeptides (aspects 4-7) having the sequences set out in figures 1-4 or degenerative variants thereof, or one or more Ka, Kb, ara-3 or ara-4 unique portion of said sequences or variants. For aspects 5-7, the preferred modes at the same time also comprised using ara-5 (see figure 5) and the analogous variants of this autoantigen.

As determined by Northern blot the atopy related autoantigens Ka, Kb, ara-3, ara-4 and ara-5 appear to be expressed in

histogenetically unrelated cells . We have also determined that they are expressed on keratinocytes and endothelial cells.

The invention is defined in the appending claims. EXPERIMENTAL PART

MATERIALS AND METHODS

IgE-immunoscreening of a human keratinocyte expression cDNA library

In a study that was unpublished when this specification was filed, we have shown that atopic dermatitis patients display IgE- autoreactivity against nitrocellulose blotted human proteins of different cell types including keratinocytes, endothelial cells, fibroblasts, platelets and mononuclear cells. The phenomenon of IgE-autoreactivity with human proteins was pronounced in atopic dermatitis patients suggesting that IgE-autoimmune mechanisms might contribute to the pathogenesis of the disease. To further characterize the IgE-autoantigens, serum IgE from an atopic dermatitis patient was used to screen a randomly primed

expression cDNA library prepared from the human keratinocyte cell line (A431).

A lambda gt 11 expression cDNA library (Clontech, Palo Alto, USA) was screened with serum IgE from a patient suffering from atopic dermatitis. In brief, 500 000 phage particles of the keratinocyte expression cDNA library were plated at a density of 20 000 particles per plate (140 mm diameter) by infecting E. coli Y1090 at 43°C (21). After plaques were visible the plates were overlaid with nitrocellulose filters (Schleicher & Schuell, Dassel, Germany), soaked in 10 mM IPTG for the induction of recombinant protein synthesis. Lambda gt 11 phage without insert were plated as well and overlaid with nitrocellulose filters for preadsorption of the patient serum before immunoscreening to reduce background reactivity of IgE with E. coli phage proteins. Serum IgE from a patient suffering from atopic dermatitis was diluted 1:10 and preincubated with nitrocellulose filters

containing E. coli lambda gt/proteins that had been incubated twice for 5 minutes and once for 30 minutes in buffer A (50 mM sodium phosphate pH 7.5, 0.5 % BSA, 0.5 % Tween 20, 0.05 % NaN₃ ) for 1 hour at 4°C. The serum was further diluted in buffer A to a final dilution of 1:20 and incubated overnight at 4°C with nitrocellulose filters containing plaquelifts of the recombinant phage. Filters were then washed as described for the blocking and incubated with 1:10 diluted ¹²⁵I-labeled anti-human IgE

(Pharmacia Diagnostics, Uppsala, Sweden) overnight at room temperature. The filters were then washed again as described for the blocking and exposed to Kodak X-OMAT S Films at -70°C using intensifying screens (Kodak, Heidelberg, Germany). Preincubation of the atopic dermatitis patient serum with E. coli/phage

proteins was found to be critical for a reduction of the

background binding of IgE to facilitate the discrimination of positive clones. Recombinant phage particles were enriched by IgE-screening to homogeneity by two rounds of recloning before preparation of phage DNA.

Characterization of cDNA clones coding for human IgE-autoantigens

More than 30 IgE-binding phage clones were obtained by the IgE- immunoscreening with serum IgE from an atopic dermatitis patient. Phage DNA was isolated using a plate lysate method and according to a restriction analysis of two clones which contained small cDNA inserts (approximately 300 base pairs) thus representing small proteins or IgE-epitopes were subcloned into plasmid pUC18. In brief, phage DNA was digested with Kpn I and Sac I to excise the cDNA inserts flanked by approximately 1000 base pairs of lambda gt 11 sequence thus allowing he cDNA to be subcloned in known orientation into plasmid pUC18. Using lambda gt 11 forward and reversed primer (Clontech, Palo Alto, USA), a sequencing kit (Pharmacia Biotech AB, Uppsala, Sweden) and ³⁵S dCTP (NEN,

Stevenhage, U.K.), the DNA sequence of both strands could be obtained (22) and allowed the determination of the orientation and correct reading frame within the β-galactosidase fusion portion. The cDNA and deduced amino acid sequence of clone Ka was compared with EMBL/SwissProt library and GenBank.

For determination of the full transcript size of the Ka and Kb mRNA the corresponding cDNA insert was isolated by Eco R I digest of the plasmid subclone. The agarose gel purified cDNA fragment was radiolabeled using 32p dCTP according to Feinberg and

Vogelstein (23) and was hybridized with nitrocellulose blotted RNA (approximately 15 μg) prepared from the human keratinocyte cell line (A431) (24) or from a human mast cell line (HMC-1) by denaturing agarose gel electrophoresis.

Expression and purification of recombinant IgE-autoantigens

The Kb IgE-autoantigen was expressed as β-galactosidase fusion protein upon infection of E. coli Y1089 with recombinant phage. Lambda gt 11 phage (negative control) without inserts were used likewise to induce synthesis of β-galactosidase which served as a control protein, β-galactosidase and the recombinant IgE

autoantigen fragments were purified using a β-galactosidase affinity matrix (Lambda ProtoSorb, Promega, Maddison, USA).

Binding of IgE antibody of atopic patients to recombinant IgE autoantigens

Purified β-galactosidase, Kb fusion protein was separated by

SDS-Page using a 8 % gel (25) and blotted to nitrocellulose (Schleicher & Schuell, Dassel, Germany) (26). Strips containing the purified proteins were blocked as described for the

plaquelifts in buffer A and incubated with diluted sera from atopic dermatitis patients, pollen allergic patients, non- allergic individuals or buffer without addition of serum. IgE- binding sera were diluted 1:10. Incubation was done as described for the plaquelifts at 4°C over night. Bound serum IgE was detected with ¹²⁵I labeled anti-human IgE antibodies (Pharmacia Diagnostics, Uppsala, Sweden).

Enhanced expression of Kb upon heat shock of cultured

keratinocytes.

The human keratinocyte cell line (A431) was incubated under lack of oxygen at 37°C and 43°C for different periods of time. Proteins were separated by SDS-PAGE and blotted to

nitrocellulose. Nitrocellulose strips were incubated with sera reacting with Kb at 55-60 kD, an atopic patient without

specificity for Kb and control sera. A buffer control without addition of serum was also included.

RESULTS AND DISCUSSION

The IgE-immune screening procedure which was used for the isolation of cDNAs coding for IgE-autoantigens was the same which had been used in earlier studies for the successful

characterization of cDNAs coding for exogenous allergens (pollen (18,27,28,29) and dog allergens (30). To reduce non-specific IgE- reactivity of the serum from atopic patients during

immunoscreening the serum was preadsorbed with E. coli/phage proteins to minimize background reactivity. Despite the elevated levels of total IgE and the remarkable IgE-reactivity of atopic dermatitis patients with E. coli proteins it was thus possible to isolate and enrich phage clones coding for human IgE- autoantigens. The cDNA inserts of two IgE-binding clones

designated Ka and Kb were excised with Kpn I and Sac I from the phage DNA together with the flanking lambda gt 11 DNA and subcloned into plasmid pUC 18. This allowed the determination of the correct reading orientation and reading frame of the cDNA within the β-galactosidase fusion protein portion. Figure 1 shows the complete cDNA and deduced amino acid sequence of the Ka cDNA and the cDNA sequence of Kb is displayed in Figure 2. The cDNA of Ka showed significant sequence identities with human cDNAs which had been isolated from skeletal muscle and hepatocyte cDNA libraries in the course of human cDNA sequencing projects (31). The identities were found by alignment of the Ka cDNA sequence with

• a partial human cDNA (GenBank accession number: Z28824; clone HSBA0F011) coding for a transcribed sequence isolated from a human skeletal muscle cDNA library during the Genexpress cDNA program, and

• a cDNA clone which was isolated from a human hepatocyte cDNA library (GenBank accession number: D12194; clone: HUM000S318; Figure 3 B)).

Both homologous cDNAs code represent incomplete fragments and no biological function of the corresponding proteins could be established as yet. It might however be assumed that the Ka cDNA and the identical homologous cDNAs code for a protein which is expressed in different cell types (muscle cells, hepatocytes, keratinocytes) and therefore might represent a rather ubiquitous and conserved protein. This corroborates our previous Western blot results showing that atopic patients display IgE reactivity to proteins of similar molecular weight present in different cell types.

To estimate the size of the complete Ka and Kb transcripts, Northern blot hybridizations were performed. The ³²P labeled Ka cDNA hybridized with RNA from human keratinocytes at

approximately 1800 nucleotides indicating that a corresponding protein of approximately 30-40 kD might be expected. Since the Ka cDNA coded only for an open reading frame of 93 amino acid residues, the polypeptide represents an IgE-epitope of the complete IgE-autoantigen against which a rather low percentage of atopic patients displayed IgE-reactivity.

The ³²P labeled Kb cDNA hybridized between the 26S and 18S RNA indicating a transcript size of approximately 2500-3000

nucleotides and a corresponding protein of approximately 50-60 kD. The cDNA coding for Kb was isolated from a keratinocyte cDNA library (A431) but apparently can be found in different cell types. By Western blotting using a rabbit anti-recombinant Kb antiserum, Kb could be detected in keratinocytes, endothelial cells and fibroblasts.

Immunoblotting of the purified Kb-β-galactosidase fusion and β- galactosidase alone after separation by 12 % SDS-PAGE showed that the Kb-fusion but not β-galactosidase could be successfully used to block IgE-binding against a protein between 50-60 kD in human keratinocytes. It was further shown that atopic dermatitis patients displayed IgE reactivity with the recombinant Kb IgE- autoantigen but not to β-galactosidase which was used as a control protein. Non-allergic individuals did not show IgE- reactivity with the recombinant IgE-epitope nor with β- galactosidase. In addition to the IgE-reactivity, binding of patients IgG to the Kb fusion protein was observed (data not shown), indicating that the interaction between Kb and human IgE and IgG antibodies reflects the interaction of the Fab part of the antibodies with the epitope. This is of particular importance in view of earlier reports regarding IgE-dependent histamine releasing factors which were assumed to interact with the

constant parts of human IgE (32,33). The occurrence of IgE- dependent histamine releasing factors has been described to be frequently associated with severe forms of atopy and atopic dermatitis, and it was concluded that these factors might be responsible for the increased capacity of basophils and mast cells of patients to release histamine without antigen

stimulation.

Our results might be interpreted that IgE-dependent histamine releasing factors could represent IgE-autoantigens which are complexed with serum IgE. Circulating IgE-autoantigen complexes might then be able to activate mast cells and basophils. Such circulating immune complexes (IgG) were already demonstrated in atopic dermatitis patients (34) and it is likely that also IgE immune complexes might occur in atopic dermatitis patients sera. Similarly as was noted for IgE-dependent histamine releasing factors we have observed that basophils from atopic patients with IgE-reactivity to human proteins showed an increased ability to spontaneously release histamine. In a final experiment it is demonstrated that the expression of Kb protein is significantly increased in stressed cells indicating that Kb might belong to a family of stress proteins.

Our results clearly prove that atopic dermatitis patients display IgE-autoreactivity to recombinant human IgE-epitopes and the working hypothesis is coined that atopic dermatitis

represents an IgE-autoimmune disease. The described recombinant IgE-autoantigens may be extremely useful for diagnosis of severe atopy and Kb might be considered as a general inflammation marker. In addition to the usefulness for diagnostic procedures the described IgE-autoantigens might be used for attempts to induce immunological tolerance in atopic patients. By applying the same methodology as described above we have, during the priority year, been able to discover three more IgE binding autoantigens (ara-3, ara-4 and ara-5) that are involved in atopy as described above. See under the legends to figure 3-5. CHARACTERISTICA OF THE DETERMINED SEQUENCES

Sequence No 1. The cDNA sequence and deduced amino acid sequence of the Ka IgE-epitope fused to β-galactosidase.

The in situ sequence of the Ka cDNA fused to β-galactosidase was determined using lambda gt 11 forward and reversed sequencing primers. The Eco R I restriction sites are printed in italics and the sequence portion coding for β-galactosidase is underlined. A 93 amino acids long open reading frame is encoded by the Ka cDNA which is terminated by a stop codon TGA indicated by an asterisk. Sequence No 2. cDNA and deduced amino acid sequence of IgE- autoantigen Kb fused to β-galactosidase.

The cDNA sequence of Kb fused to the sequence coding for β- galactosidase (underlined) is displayed. The deduced amino acid sequence is displayed below the nucleotide sequence. The Eco R I restriction sites are printed in italics. Due to an Eco R I linker dimer the open reading frame comprising 1347 nucleotides is fused in frame to β-galactosidase. Both strands of the cDNA sequence were determined according to the method of Sanger. An internal Sac I restriction site (GAGCTC) could be found starting with nucleotide 1202. No start signal (ATG) was present at the 5' end of the cDNA indicating that the cDNA clone is incomplete.

Sequence No 3. Nucleotide sequence of ara-3.

This insert contained 1501 bp. A comparison with the GenBank at NIH showed that the sequence was homologous at the 5' end with cDNA clones derived from the human gall-bladder and leg muscle, and at the 3' end with a second cDNAs clone derived from leg muscle. No biological function has yet been found for these proteins. Sequence No 4. Nucleotide sequence of ara-4.

This clone has two internal SacI and one EcoRI restriction sites. The phage insert contains 1700 bp. A comparison with known DNA sequences showed that the sequence was homologous at the 5' end with a cDNA clone from a human brain (infant with muscle atrophy) and at the 3' end with different cDNA clones derived from human tissues (brain of a healthy infant (Khan et al.,

1992), keratinocytes (one clone) and unknown tissue (two clones). The biological function of the corresponding proteins have so far not been determined.

Sequence No 5. Nucleotide sequence of ara-5.

This clone was 900 bp and contained an internal SacI

restriction site. 500 bp were sequenced. The sequenced part was homologous to human keratin type II.

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Claims

P A T E N T C L A I M S

1. Recombinant DNA molecule comprising

i) a nucleotide sequence which codes

i:3) for a peptide comprising at least one epitope unique for (a) these atopy related antigens or for (b) a peptide that is homologous to > 50 %, such as > 60 %, > 70 % or > 80 % to the Ka or Kb antigen, or ii) a nucleotide sequence which hybridizes with such a

sequence under conditions of high stringency.

2. Recombinant DNA molecule according to claim 1, comprising at last one of the nucleotide sequences set out in figures 1-4, respectively, or degenerative variants thereof, or one or more Ka, Kb, ara-3 or ara-4 unique epitopes of said sequences or variants.

3. Recombinant DNA expression vector or cloning system

comprising an expression control sequence operatively linked to a nucleotide sequence coding a peptide and defined in (i) and (ii) of claim 1 or 2.

4. Host cell or host system containing a recombinant DNA

molecule or a recombinant expression vector as defined in any of claims 1-3.

5. Polypeptide comprising at least one of the amino acid

sequence defined by the nucleotide sequences coding for the atopy related antigens Ka, Kb, ara-3 and ara-4, or at least one epitope unique for these atopy related antigens.

6. Polypeptide according to claim 5 wherein the polypeptide has been recombinantly or synthetically produced.

7. Polypeptide according to any of claims 5-6 comprising at

least one of the amino acid sequences encoded by the

nucleotide sequences set out in figures 1-4, respectively, or an epitope thereof unique for the anyone of the atopy related antigens Ka, Kb, ara-3 or ara-4.

8. Polypeptide according to any of claims 5-7 wherein the

peptide is derivatized in the sense that it is a) linked to a water-insoluble phase by physical adsorption or by covalent bonding, b) conjugated covalently to an analytically

detectable group (label), or c) covalently linked to an additionally polypeptide.

9. Polypeptide according to claim 8 wherein it is recombinantly produced in the form of a fusion protein comprising the additional protein.

10. Polypeptide according to claim 9 wherein said additional

polypeptide is β-galactosidase, GST or lambda ell protein or any other polypeptide which can be expressed in a prokaryotic or an eukaryotic cell.

11. Polypeptide according claim 8 wherein the water-insoluble

phase is a water-insoluble polymer that may have a physical form selected from: walls of microtitre wells, spheres, rods, sheets, strips, pads etc, said physical forms may be porous or non-porous.

12. Polypeptide according claim 8 wherein the water-insoluble

phase is a water-insoluble polymer selected from water- insoluble forms of polysaccharides and their derivatives, for instance dextran, pullulan, agarose, cellulose etc, or synthetic polymers, preferably vinyl polymers, such as polyacrylamides, polyacrylates, polystyrene, polyvinyl alcohol etc.

13. Method for in vitro diagnosis of atopy or inflammation in an a mammalian individual, often a human individual, which method comprises detection of the reaction of IgE in a body fluid sample from the individual with an IgE autoantigen from the same species as the individual or an IgE

hapten/autoantigen crossreacting with the IgE autoantigen, optionally derived from another source, wherein the formation of an IgE immune complex is taken as an indication that the individual is suffering from atopy, in particular atopic dermatitis or asthma bronchiale, or inflammation.

14. Method according to claim 11 wherein the autoantigen is

according to any of claims 5-12 or ara-5 as definied in figure 5 including an IgE epitope of ara-5.

15. Method according to any of claims 13-14 wherein the body

fluid sample is derived from blood, such as a whole blood sample, a serum sample or a plasma sample.

16. Method according to any of claims 13-15 wherein the

autoantigen in insoluble form or in insolubilizable form is brought into contact with the sample under conditions permitting formation of an immune complex between the autoantigen and antibodies present in the sample, whereafter the amount of complexed IgE antibodies is detected, where appropriate after insolubilization of the formed IgE- autoantigen complex.

17. Method according to any of claims 13-15 wherein the

autoantigen is soluble and labeled with an analytically detectable group, and the immune complex between the

autoantigen and sample IgE is formed in a soluble form that in the course of being quantitated is insolubilized by being contacted with water-insoluble or water-insolubilizable anti- IgE antibodies, preferably linked to the solid phases given in claim 12.

18. Method to measure in vitro the cellular reaction against an IgE-autoantigen, in which a polypeptide according to any of claims 5-12 is used to stimulate or to inhibit the cellular reaction.

19. Method of treatment of an mammalian individual, in particular a human being, suffering from atopy wherein an effective amount of a polypeptide according to any of claims 5-12 is administered to said individual.

20. Method of diagnosing atopy or inflammation, which comprises measuring an atopy related antigen as defined in claims 5-12, or an antibody, preferably of IgE class.